2021-2023年間台灣家禽白血病病毒的分離與序列分析及利用RCAS重組本土病毒株研究傳播特性

Abstract

目前影響全世界家禽的反轉錄病毒主要為家禽白血病病毒（avian leukosis virus, ALV）和網狀內皮增生病毒（reticuloendotheliosis virus, REV），其中ALV會引起淋巴球性白血病（lymphoid leukosis, LL）和骨髓球性白血病（myeloid leukosis, ML）等腫瘤性疾病，造成重大經濟損失。根據其膜蛋白之gp85，ALV被分為A到K亞群，其中ALV-J最為盛行，尤其是在中國等地。由於內源性ALV的存在、ALV的高突變率以及家禽複雜的產業生態等因素，要從雞群中消除ALV是一件非常艱鉅的任務。而RCAS（replication competent ALV LTR with a splice acceptor）載體系統，源自同時也是一種ALV的Rous sarcoma virus（RSV），對研究ALV病毒與宿主相互作用至關重要。RCAS載體能將GFP或mCherry等基因導入生物體中，有助於觀察ALV的感染路徑和複製狀況。本研究希望能瞭解目前ALV在台灣雞場中的感染情況，並使用本地的ALV分離株建立一個RCAS重組病毒，應用於實驗中研究ALV的傳播途徑。在2021-2023年間，ALV在8個雞場中被檢測陽性，其中ALV-J最為普遍（8／8），其次是ALV-K（3／8）和ALV-A（1／8），其中也在3個雞場中觀察到了不同ALV亞群的共同感染。由gp85的序列分析顯示，台灣以往和近期的ALV病毒株在親緣樹上分佈在多個分支上。本研究使用DF-1細胞分離出了5種ALV-J病毒株Av21-01、Av23-03、Av23-25(J)、Av23-30(J)、Av23-33(J)和1種ALV-K病毒株Av23-30(K)，經由分析gp85和3UTR的序列，彌補了台灣對ALV-J和ALV-K調查超過十年的空窗期。同時，利用Av21-01 env替換了RCAS的env，創建了一種能在DF-1細胞中複製並表現綠色螢光訊號的重組病毒，並進一步探討了其潛在的致病性、感染路徑以及其在小雞的分佈途徑。當替換的序列不同時，也對重組病毒在DF-1細胞中的複製能力產生了影響。此次研究結果證實了ALV在台灣雞場中仍持續存在且流行，並建立了含本土病毒株基因序列的重組RCAS病毒，此重組病毒未來將應用於研究病毒傳播途徑及入侵機制，以期望能幫助台灣家禽產業控制ALV-J。

Avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) are the major retroviruses impacting poultry globally, causing neoplastic diseases like lymphoid leukosis (LL) and myeloid leukosis (ML), leading to significant economic losses. ALVs are categorized into subgroups A to K based on their envelope glycoprotein (gp85), with ALV-J being the most prevalent worldwide, especially in regions such as China. Eliminating ALV from flocks is challenging due to factors such as endogenous viruses, high mutation rates, and diverse poultry-producing types. The RCAS vector system, derived from Rous sarcoma virus (RSV), which also belongs to ALV, has been crucial in studying ALV virus-host interactions. RCAS (replication competent ALV LTR with a splice acceptor) vectors enable the introduction of genes like GFP or mCherry into organisms, aiding in observing ALV infection routes and replication status. This study aimed to assess ALV infection in chicken farms in Taiwan and establish a reporter system using a local ALV isolate. ALV was detected in 8 chicken farms between 2021 and 2023, with ALV-J being the most prevalent (8/8), followed by ALV-K (3/8) and ALV-A (1/8). Co-infections of different ALV subgroups were also observed in three farms. Sequence analysis of gp85 showed that previous and recent ALV strains in Taiwan were distributed in multiple clusters on the phylogenetic tree. Notably, five ALV-J strains Av21-01, Av23-03, Av23-25(J), Av23-30(J), Av23-33(J) and one ALV-K strain Av23-30(K) were isolated using DF-1 cells. Analysis of the gp85 and 3UTR sequences of these virus strains filled the gap in the investigation of ALV-J and ALV-K in Taiwan for over a decade. Meanwhile, by substituting the RCAS env with the Av21-01 env, a recombinant virus was created that replicates in DF-1 cells and expresses green fluorescent signals. Further experiments were conducted on its potential pathogenicity, infection routes, and distribution in chicks. It was observed that substituting sequences beyond just the envelope gene affected the replication ability of the recombinant virus in DF-1 cells. This study elucidated the ongoing prevalence and persistence of ALV in chicken farms in Taiwan and established a recombinant RCAS virus containing the gene sequences of a local strain. This recombinant virus will be used in future studies to explore virus transmission pathways and invasion mechanisms, aiming to help the Taiwanese poultry industry control ALV-J.